Project Summary/Abstract It is estimated that thousands of diseases are caused by genetic mutation and subsequent aberrant protein localization within the cell, though pathological mechanisms are understood for only a fraction of all known mutations. Here we propose to engineer a widely-applicable platform which can visualize real-time protein trafficking events in living cells. First, we will rationally design a mutant library of Saccharomyces cerevisiae tryptophanyl-tRNA synthetases which will recognize a small coumarin fluorescent amino acid (ScCoumarin-RS). The S. cerevisiae enzyme is thought to be orthogonal to human tryptophan aminoacylation processes, yet still recognizable to the mammalian cell's translational machinery including elongation factors and the ribosome complex. We will also engineer a cognate S. cerevisiae amber suppressor coumarinyl tRNA, which will be charged with free coumarin by the ScCoumarin-RS. When cloned into a mammalian vector, these engineered biomolecules will insert the small coumarin fluorescent amino acid into a desired position within the polypeptide chain of the protein without perturbing its native structure or function. After confirming in cellulo functionality using a GFP reporter assay, we will apply the technology to a proof-of-concept mistrafficking cellular disease model of Lri-Weill dyschondrosteosis (LWD), a rare form of skeletal dysplasia caused by a genetic mutation in the SHOX gene. A single missense mutation abolishes the nuclear localization signal within the SHOXa transcription factor, and thus it accumulates in the cytoplasm instead of translocating to the nucleus. Our platform will visualize both the wild-type protein nuclear trafficking, as well as the mutant protein cytosolic localization. We will also quantify the kinetics of protein accumulation over an 18-hour time course. This novel platform technology has broad applicability to protein localization studies which can reveal the pathogenesis of mistrafficking pathways, validate suspected mistrafficking mutations, and enable a greater understanding of disease-related phenotypes at the subcellular level. The proposed project will allow two graduate students and two undergraduate students to perform research in the biomedical field. Many of these students are following a pre-med track coursework and would benefit from this unique opportunity to work at the leading edge of biomedical research, inspiring them to pursue biomedical careers.